Results and Discussion
Several studies have reported and confirmed infections byFusarium pathogens in sea turtles around the world and have investigated the relationship between FSSC infections and hatching success (e.g. Sarmiento-Ramirez et al., 2010; 2014). However, the negative effects of FSSC in freshwater turtles have not been reported until now. We found that all 23 nests of the yellow-spotted turtle monitored in this study had eggs showing symptoms of fusariosis, with an average of 42% of eggs symptomatic across nests (range 10-100%; Table S2). There was significant variation in the proportion of diseased eggs between nests (X21, 22= 192.48; p-value < 0.001; adj-R2 = 0.332) suggesting that some nests were more vulnerable to infection than others. Symptoms of FSSC infections in eggs of the yellow-spotted turtle were significantly associated with hatching success (X21, 677= 149.86; p-value < 0.001; adj-R2 = 0.525) with a 72% hatching success for asymptomatic eggs (n = 394), whilst only 8% of symptomatic eggs hatched (n = 286) (Figure 1b). In line with our results, experimental inoculation of F. solani in eggs of the sea turtleCaretta caretta resulted in a hatching success rate of 18% (n = 12) compared to 92% (n = 12) for uninfected controls (Sarmiento-Ramirez et al., 2010). Similar disparity in hatching rates according to symptoms of fusariosis were also observed in the natural environment in endangered sea turtles, with up to 92% embryo mortality in diseased eggs (Sarmiento-Ramirez et al., 2014).
Fusarium -specific PCR tests of 29 asymptomatic and 39 symptomatic eggs revealed that 59% of the symptomatic eggs tested positive forFusarium (Table S3), whilst the remaining 41% tested negative (Table S3), suggesting that infected eggs may not only be afflicted by pathogenic Fusarium and that there are multiple causes of infection. Indeed, previous studies have reported infections of sea-turtle eggs caused by different fungi within Aspergillus ,Rhizopus and Apophysomyces (Candan, 2018; Santos Costa-Neves et al., 2015). Interestingly, we also found that 28% of asymptomatic eggs tested positive for Fusarium (Table S3), suggesting either that these eggs were at an initial stage of infection or that colonization ofFusarium did not lead to the onset of pathogenic fusariosis. Indeed, although the pathogenicity of F. solani in sea turtles has been demonstrated, it is not well understood if colonization is systematically pathogenic and other species of Fusarium may not be pathogenic (Sarmiento-Ramirez et al., 2010).
From our sequencing of amplicons (from three asymptomatic and four symptomatic eggs), we obtained nine Fusarium amplicon sequence variants (ASVs) belonging to the pathogenic FSSC (PP= 1; Figure 2). From the obtained ASVs, ASV_1 and ASV_3 were present in both asymptomatic and symptomatic eggs, ASV_4 and ASV_5 were each present in two different symptomatic eggs, and the remaining five ASVs were only present in asymptomatic eggs (Table S4). ASV_3 and ASV_6 grouped withF. keratoplasticum and F.solani isolates from human, crops and sea turtle egg infections. ASV_4 grouped with F. solaniisolated from crops and P. unifilis eggs (PP= 0.98; Figure 2). ASV_2, _5, and _7 grouped with F. falciforme isolates from the environment, crops, human, and sea turtle egg infections (PP= 0.915; Figure 2). In contrast, ASV_1, _8 and _9 grouped together with sixF. keratoplasticum isolates from P. unifilis eggs (PP= 0.933; Figure 2). Our results add novel data to a recent study reporting the presence of F. keratoplasticum in P. unilfilis eggs in artificial nests of the same geographical region (García-Martín et al., 2021) by adding the incidence of the FSSC’s F. falciforme andF.solani .
Although both our study and that of García-Martín et al. (2021) were conducted on harvested eggs, the source of pathogenic FSSC is likely to be vertical transmission from the mother or horizontal transmission from the natural environment (since eggs are incubated in sand from the original nesting site). Whilst it is possible that infection may spread more easily within artificial nests than in natural settings, our combined results suggest a possible Fusarium infection outbreak in a remote area of the Amazon and that it is likely to be a major contributor of infectious hatching failure in this freshwater turtle species. This has important implications for turtle conservation efforts worldwide, since egg harvesting, and artificial incubation is a major strategy applied by turtle conservationists. Further studies are necessary to understand the epidemiology and distribution ofFusarium pathogenic fungi causing hatching failure of endangered non-marine turtle species worldwide.